Methods for large scale production of recombinant DNA-derived tPA or K2S molecules

ABSTRACT

The invention belongs to the field of thrombolysis and of tissue plasminogen activator (tPA) derivative production in prokaryotic cells. The invention relates to methods for the production of a recombinant DNA-derived tPA, a variant thereof or a (Kringle 2 Serine) K2S molecule or a variant thereof in prokaryotic cells, wherein the tPA or K2S or variant is secreted extracellularly as an active and correctly folded protein, and the prokaryotic cell contains and expresses a vector comprising the DNA coding for the tPA or K2S or variant operably linked to the DNA coding for the signal peptide OmpA. The invention further relates to specific K2S derivatives obtainable by the method. The invention further relates to the DNA molecules and the use of the DNA molecules in the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application .Ser. No.09/987,455, filed Nov. 14, 2001, now U.S. Pat. No. 6,955,910, whichclaims the benefit of U.S. Provisional Patent Application No.60/268,574, filed Feb. 15, 2001, and claims the benefit of Great BritainPatent Application No. GB 0027779.8, filed Nov. 14, 2000. The fulldisclosure of each of these applications is herein incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention belongs to the field of thrombolysis and of tissueplasminogen activator (tPA) derivative production in prokaryotic cells.

The invention relates to methods for the production of a recombinantDNA-derived tPA, a variant thereof or a (Kringle 2 Serine) K2S moleculeor a variant thereof in prokaryotic cells, wherein said tPA or K2S orvariant is secreted extracellularly as an active and correctly foldedprotein, and the prokaryotic cell contains and expresses a vectorcomprising the DNA coding for said tPA or K2S or variant operably linkedto the DNA coding for the signal peptide OmpA. The invention furtherrelates to specific K2S derivatives obtainable by said method. Theinvention further relates to said DNA molecules and the use of said DNAmolecules in said methods.

2. Related Art

Tissue plasminogen activator (tPA) is a polypeptide containing 527 aminoacid residues (Pennica, D., et al., Nature 301:214-221 (1983)) with amolecular mass of 72 kDa. The molecule is divided into five structuraldomains. Nearby the N-terminal region is a looped finger domain, whichis followed by a growth factor domain. Two similar domains, kringle 1and kringle 2, are following. Both finger and kringle 2 domains bindspecifically to the fibrin clots thereby accelerating tPA proteinactivation of bound plasminogen. Downstream of kringle 2 is the serineprotease, with its catalytic site located at the C-terminus. The serineprotease is responsible for converting plasminogen to plasmin a reactionimportant in the homeostasis of fibrin formation and clot dissolution.The correct folding of tPA requires the correct pairing of 17 disulfidebridges in the molecule (Allen, S., et al., J. Biol. Chem. 270:4797-4804(1995)).

Clinically, tPA is a thrombolytic agent of choice for the treatment ofacute myocardial infarction, pulmonary embolism, stroke, peripheralarterial occlusions, and other thromboembolic diseases. It has theadvantage of causing no side effects on systemic hemorrhaging andfibrinogen depletion (Camiolo, S. M., et al., Proc. Soc. Exp. Biol. Med.38:277-280 (1971)). Bowes melanoma cells were first used as a source intPA production for therapeutic purposes (Griffiths, J. B. andElectricwala, A., Adv. Biochem. Eng. Biotechnol. 34:147-166 (1987)).Since a consistent process with efficient production of highly purifiedprotein in good yield is required for clinical use, the construction offull-length recombinant-tPA (r-tPA) progressed to mammalian cells.Chinese hamster ovary cells were transfected with the tPA gene tosynthesize the r-tPA (Cartwright, T., “Production of t-PA from animalcell culture,” in Animal Cell Biotechnology, Vol 5, Spier and Griffithseds., Academic Press, New York, N.Y. (1992), pp 217-245; Lubiniecki, A.,et al., “Selected strategies for manufacture and control of recombinanttissue plasminogen activator prepared from cell culture,” in Spier, etal., eds., Advances In Animal Cell Biology And Technology ForBioprocesses, Butterworths, London, p. 442-451). The recombinant DNAderived product produced by a mammalian cell culture fermentation systemis harvested and purified from the culture medium. Attracted bysimplicity and economy of production, a number of efforts in producingr-tPA from microorganisms, especially bacteria, and more especially fromEscherichia coli, were investigated (Datar, R. V., et al., Biotechnology11:349-357 (1993); Harris, T. J., et al., Mol. Biol. Med. 3:279-292(1986); Sarmientos, P., et al., Biotechnology 7:495-501 (1989)).Regarding the low yield and the formation of inclusion bodies, whichresulted in misfolding and in an inactive enzyme, numerous strategieshave been proposed to overcome these problems.

Several deletion-mutant variants including kringle 2 plus serineprotease (K2S) were considered. However, the enzymatic activity of therecombinant-K2S (r-K2S) was obtained only when refolding processes ofpurified inclusion bodies from cytoplasmic compartment were achieved(Hu, C. K., et al., Biochemistry 33:11760-11766 (1994); Saito, Y., etal., Biotechnol. Prog. 10:472-479 (1994)). In order to avoid thecumbersome refolding processes, impurities of misfolded proteins, andperiplasmic protein delivery, special bacterial expression systems wereexploited (Betton, J. M., et al., J. Biol. Chem. 273:8897-8902 (1998);Scherrer, S., et al., Appl. Microbiol. Biotechnol. 42:85-89 (1994)).Despite periplasmic expression of tPA, overexpression led to inactiveaggregates, even in the relatively high oxidizing condition in theperiplasm.

In the prior art, there are a few descriptions of methods for thepreparation of recombinant K2S in E. coli. However, there is nodisclosure of a method leading to a cost effective method for largescale production of biologically active K2S.

Obukowicz et al. (Obukowicz, M. G., et al., Biochemistry 29:9737-9745(1990)) expressed and purified r-K2S from periplasmic space. The obviousdisadvantage of this method was an extra periplasmic extraction step,which is not suitable for large scale production.

Saito et al. (Saito, Y., et al., Biotechnol. Prog. 10:472-479 (1994))disclose the cytoplasmic expression of r-K2S. The authors used an invivo renaturation processes for the expressed r-K2S, which was purifiedfrom the cytoplasmic space of E. coli as inclusion body. BoehringerMannheim use a similar cumbersome denaturing/refolding process involvingthe steps of cell digestion, solubilization under denaturing andreducing conditions and reactivation under oxidizing conditions in thepresence of GSH/GSSG which is not cost effective (Martin, U., et al.,Kardiol. 79:167-170 (1990)) and requires mutation of the amino acidsequence with possibly antigenic potential.

In 1991, Waldenström et al. (Waldenström, M., et al., Gene 99:243-248(1991)) constructed a vector (pEZZK2P) for the secretion of kringle 2plus serine protease domain to E. coli culture supernatant.Hydroxylamine was used to remove the ZZ fusion peptide fromIgG-Sepharose purified fraction. The cleavage agent hydroxylaminerequired modification of the cleavage sites of kringle 2 plus serineprotease (Asn177→Ser and Asn184→Gln) thus to protect it fromhydroxylamine digestion. However, the resulting non-native, not properlyfolded K2S molecule is not suitable for therapeutic purposes. Noenzymatic activity regarding fibrin binding/protease activity wasdisclosed. The unusual sequence may even activate the human immunesystem.

SUMMARY OF THE INVENTION

The problem underlying the present invention was thus to provide acommercially applicable method for large scale production of tPAmolecules and derivatives thereof, e.g. K2S, wherein the K2S molecule issecreted in its biologically active form into the culture supernatant.The problem was solved within the scope of the claims and specificationof the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Validation of PCR amplification product of the K2S gene from thep51-3 vector by using SK2/174 and ASSP primers. Lane 1 shows 1 kb marker(Roche Molecular Biochemicals, Indianapolis, Ind.). Lane 2 was loadedwith 1 μl of amplified product. A single band at 1110 bp is depicted.The electrophoresis was performed on a 1% agarose gel.

FIG. 2. Identification of inserted K2S gene at 1110 bp (*) after Sfi Idigested pComb3H-K2S was demonstrated in lane 3. Lane 1 shows 1 kbmarker. Lane 2 was loaded with uncut pComb3H-K2S. The electrophoresiswas performed on a 1% agarose gel.

FIG. 3. Scheme of pComb3H-K2S showing two Sfi I cloning sites into whichthe K2S gene was inserted. Signal sequence (OmpA), ribosome binding site(RIBS), lac promoter, and gpIII gene are also depicted.

FIG. 4. Schematic diagram of the mutation site at the junction betweenthe K2S and gpIII genes on pComb3H-K2S. The annealing site ofpComb3H-K2S is bound with a set of mutation primers (MSTPA and MASTPA)containing modified oligonucleosides (underlined). After performing thecycle amplification, the Sfi I site 1 (in bold) is modified and lost inthe newly synthesized strand.

FIG. 5. Characterization of newly synthesized MpComb3H-K2S by the Sfi Irestriction enzyme. A single band at 4319 bp that refers to a singlecleavage site of MpComb3H-K2S is observed in lane 3. No inserted K2Sband at 1110 bp can be visualized. Lane 1 shows 1 kb marker. Lane 2 wasloaded with uncut MpComb3H-K2S. The electrophoresis was performed on a1% agarose gel.

FIG. 6. Identification of immunological reactive band with ofrecombinant DNA-derived protein purified from XM[K2S] culturesupernatant with sheep anti-tPA conjugated HRP. Lane 1 was loaded with40 ng of standard melanoma tPA (86/670), which showed the reactive bandat 70 kDa. The partially purified and concentrated culture supernatantsfrom non-transformed E. coli XL1-Blue and XM[K2S] were applied to lane 2and 3 respectively. The distinct reactive band was particularlydemonstrated in lane 3 at 39 kDa.

FIG. 7. Molecular weight determination of extracellular r-K2S harboringactive serine protease domain by copolymerized plasminogenpolyacrylamide gel electrophoresis. Lane 1 contained the indicatedmolecular weight standards (×10⁻³), SDS-6H (Sigma, Saint Louis, Mo.).Fifty μg of the 55% saturated ammonium sulfate precipitated culturesupernatant of XL-1 Blue, Xl-1 Blue transformed with pComb3HSS, andXM[K2S] were loaded in lane 2, 3, and 4 respectively. Lane 5 contained50 mIU of standard melanoma tPA (86/670). Transparent zones of digestedplasminogen in polyacrylamide gel are visible only in lane 4 atmolecular weight of 34 and 37 kDa (B) and lane 5 at molecular weight of66 and 72 kDa (A).

FIG. 8. Structure A (SEQ ID NO:11) Native K2S molecule from amino acids174-527 without modification.

FIG. 9. Structure B-0 (SEQ ID NO:12) Native K2S molecule from aminoacids 197-527 without modification.

FIG. 10. Structure B-1 (SEQ ID NO:13) K2S molecule from amino acids193-527, wherein to Structure B-0 of FIG. 9 the amino acids SEGN wereadded at the N-terminal portion.

FIG. 11. Structure B-2 (SEQ ID NO:14) K2S molecule from amino acids193-527, as in FIG. 10, wherein Cys-261 was exchanged for Ser.

FIG. 12. Structure B-3 (SEQ ID NO:15) K2S molecule from amino acids191-527, wherein to Structure B-0 of FIG. 9 the amino acids SEGNSD wereadded at the N-terminal portion.

FIG. 13. Structure B-4 (SEQ ID NO:16) K2S molecule from amino acids191-527, as in FIG. 12, wherein Cys-261 was exchanged for Ser.

FIG. 14. Structure C (SEQ ID NO:17) Native K2S molecule from amino acids220-527 without modification. This molecule may be further modified in asimilar manner as disclosed for structure B in FIGS. 10-13.

FIG. 15. Structure D (SEQ ID NO:18) Native K2S molecule from amino acids260-527 without modification. This molecule may be further modified in asimilar manner as disclosed for structure B in FIGS. 10-13.

FIG. 16. tPA molecule (SEQ ID NO:19).

TABLE 1 DETECTION OF R-K2S MOLECULE IN PHAGE PREPARATION BY SANDWICHELISA Tracer antibody (conjugated HRP) Capture Anti-tPA Anti-M13antibody K2S-Φ VCSM13^(a) K2S-Φ VCSM13 Anti-kringle 2^(b) 1.12 ±0.04^(c) 0.12 ± 0.03 1.89 ± 0.02 0.16 ± 0.02 Anti-M13 0.17 ± 0.01 0.14 ±0.05 1.91 ± 0.02 1.88 ± 0.03 ^(a)VCSM13 was harvested from XL-1 Bluetransformed with pComb3HSS. ^(b)Mouse monoclonal anti-kringle 2 (16/B)was used. The other antibodies were prepared from sheep immunoglobulin.^(c)Value is mean of absorbance of each sample which was assayed intriplicate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The use of the singular or plural in the claims or specification is inno way intended to be limiting and also includes the other form.

The invention relates to a method for the production of a recombinantDNA-derived tissue plasminogen activator (tPA), a tPA variant, a Kringle2 Serine protease molecule (K2S) or a K2S variant in prokaryotic cells,wherein said tPA, tPA variant, K2S molecule or K2S variant is secretedextracellularly as an active and correctly folded protein, characterizedin that the prokaryotic cell contains and expresses a vector comprisingthe DNA coding for said tPA, tPA variant, K2S molecule or K2S variantoperably linked to the DNA coding for the signal peptide OmpA or afunctional derivative thereof.

Surprisingly, the use of the signal peptide OmpA (SEQ ID NO:3) aloneand/or in combination with the N-terminal amino acids SEGN (SEQ ID NO:9)/SEGNSD (SEQ ID NO: 10) translocate the recombinant DNA-derived tPA,tPA variant, K2S molecule or K2S variant to the outer surface andfacilitates the release of the functional and active molecule into theculture medium to a greater extent than any other method in the priorart. Before crossing the outer membrane, the recombinant DNA-derivedprotein is correctly folded according to the method of the presentinvention. The signal peptide is cleaved off to produce a maturemolecule. Surprisingly, the efficiency of signal peptide removal is veryhigh and leads to correct folding of the recombinant DNA-derivedprotein.

Said signal peptide OmpA interacts with SecE and is delivered across theinner membrane by energy generated by SecA, which binds to Seccomponents (SecE-SecY). SecY forms a secretion pore to dispatch therecombinant DNA-derived protein according to the invention. The spacebetween the outer membrane and inner membrane of Gram-negative bacteria,periplasm, has higher oxidative condition in comparison to thecytoplasmic space. This supports the formation of disulfide bonds andproperly folding of the recombinant DNA-derived protein (e.g. K2S) inthe periplasm to yield an active molecule. According to the presentinvention, the signal peptide will be cleaved off to produce a maturemolecule. The complex of GspD secretin and GspS lipoprotein on the outermembrane serves as gate channel for secreting the recombinantDNA-derived protein according to the invention to the extracellularmedium. This secretion process requires energy, which is generated incytoplasm by GspE nucleotide-binding protein then transferred to theinner membrane protein (Gsp G-J, F and K-N). GspC transfers the energyto GspD by forming a cross-linker between a set of inner membraneprotein (Gsp G-J, F and K-N) and GspD. Before crossing the outermembrane successfully, the recombinant DNA-derived protein is correctlyfolded.

Operably linked according to the invention means that the DNA encodingthe tPA, tPA variant, K2S molecule or K2S variant (preferably comprisingthe nucleic acid encoding SEGN (SEQ ID NO:9) or SEGNSD (SEQ ID NO: 10)at its N-terminal portion) is cloned in close proximity to the OmpA DNAinto the vector in order to achieve expression of the OmpA-tPA, tPAvariant, K2S molecule or K2S variant-fusion protein and to directsecretion outside the prokaryotic host cell. Typically, the majority ofthe tPA, tPA variant, K2S molecule or K2S variant is secreted and canthen be purified by appropriate methods such as ammonium sulfateprecipitation and/or affinity chromatography and further purificationsteps. The invention also includes the use of inducers such as IPTG orIPTG in combination with glycerol, the improvement of the incubationcondition and harvesting period to maximize the amount of activeprotein.

In a preferred embodiment, said DNA encoding the OmpA signal peptide maybe fused to a short peptide characterized by the amino acid sequenceSEGN (SEQ ID NO:9) or SEGNSD (SEQ ID NO: 10) or the coding nucleic acidsequence TCTGAGGGAAAC (SEQ ID NO:20) or TCTGAGGGAAACAGTGAC (SEQ ID NO:1)and located in the N-terminal portion or at the N-terminal portion ofthe tPA, tPA variant, K2S molecule or K2S variant. Thus, preferably,said fusion protein comprises OmpA-SEGNSD-tPA, -tPA-variant,-K2S-molecule or -K2S-variant. Even more preferred, said amino acidscharacterized by SEGN or SEGNSD may be carry a point mutation or may besubstituted by a non-natural amino acid. Even more preferred, there maybe an amino acid or non-amino acid spacer between OmpA and SEGN orSEGNSD and the tPA, tPA variant, K2S molecule or K2S variant.

Thus, in a preferred method according to the invention said theprokaryotic cell contains and expresses a vector comprising the DNAcoding for said tPA, tPA variant, K2S molecule or K2S variant operablylinked to the DNA coding for the signal peptide OmpA which is operablylinked to the nucleic acid molecule defined by the sequenceTCTGAGGGAAACAGTGAC (SEQ ID NO: 1) or a functional derivative thereof.

The method according to the invention comprises prokaryotic host cellssuch as, but not limited to Escherichia coli (E. coli), Bacillussubtilis, Streptomyces, Pseudomonas, e.g. Pseudomonas putida, Proteusmirabilis, Saccharomyces, Pichia or Staphylococcus, e.g. Staphylococcuscarnosus. Preferably said host cells according to the invention areGram-negative bacteria.

Preferably, a method according to the invention is also characterised inthat the prokaryotic cell is E. coli. Suitable strains include, but arenot limited to E. coli XL-1 blue, BL21(DE3), JM109, DH series, TOP10 andHB101. Preferably, a method according to the invention is alsocharacterised in that the following steps are carried out:

-   -   a) the DNA encoding the tPA, tPA variant, K2S molecule or K2S        variant is amplified by PCR;    -   b) the PCR product is purified;    -   c) said PCR product is inserted into a vector comprising the DNA        coding for OmpA signal peptide and the DNA coding for gpIII in        such a way that said PCR product is operably linked upstream to        the DNA coding for the OmpA signal sequence and linked        downstream to the DNA coding for gpIII of said vector;    -   d) that a stop codon is inserted between said tPA, tPA variant,        K2S molecule or K2S variant and gpIII;    -   e) said vector is expressed by the prokaryotic cell    -   f) the tPA, tPA variant, K2S molecule or K2S variant is        purified.

For step a) according to the invention the choice/design of the primersis important to clone the DNA in the right location and direction of theexpression vector (see example 1). Thus, the primers as exemplified inexample 1 and FIG. 4 comprise an important aspect of the presentinvention. With gp III of step c) gene protein III is meant which ispresent mainly in phagemid vectors. The stop codon is inserted to avoidtranscription of gp III thus eventually leading to secretion of the tPA,tPA variant, K2S molecule or K2S variant of interest. Any suitablemethod for insertion of the stop codon may be employed such assite-directed mutagenesis (e.g., Weiner M P, Costa G L (1994) PCRMethods Appl 4(3):S131-136; Weiner M P, Costa G L, Schoettlin W, ClineJ, Mathur E, Bauer J C (1994) Gene 151(1-2):119-123; see also example1).

Any vector may be used in the method according to the invention,preferably said vector is a phagemid vector (see below).

Preferably, a method according to the invention is also characterised inthat the tPA, tPA variant, K2S molecule or K2S variant is selected fromhuman tissue plasminogen activator (tPA, FIG. 16) or a fragment, afunctional variant, an allelic variant, a subunit, a chemicalderivative, a fusion protein or a glycosylation variant thereof. Suchfragments, allelic variants, functional variants, variants based on thedegenerative nucleic acid code, fusion proteins with an tPA proteinaccording to the invention, chemical derivatives or a glycosylationvariant of the tPA proteins according to the invention may include one,several or all of the following domains or subunits or variants thereof:

-   -   1. Finger domain (4-50)    -   2. Growth factor domain (50-87)    -   3. Kringle 1 domain (87-176)    -   4. Kringle 2 domain (176-262)    -   5. Protease domain (276-527)

The numbering/naming of the domains is according to Genbank accessionnumber GI 137119 or Nature 301 (5897), 214-221 (1983).

More preferably, a method according to the invention is alsocharacterised in that the tPA, tPA variant, K2S molecule or K2S variantis selected from the Kringle 2 (Barbas, C. F. III and Wagner, J., etal., Enzymology 8: 94-103 (1995)) plus Serine protease (Bennett, W. F.,et al., J. Biol. Chem. 266:5191-5201 (1991)) K2S variant of human tissueplasminogen activator or a fragment, a functional variant, an allelicvariant, a subunit, a chemical derivative, a fusion protein or aglycosylation variant thereof.

More preferably, a method according to the invention is alsocharacterised in that the vector is a phagemid vector comprising the DNAcoding for OmpA signal peptide and the DNA coding for gpIII.

More preferably, a method according to the invention is alsocharacterised in that the vector is the pComb3HSS phagemid (see alsoexample 1).

More preferably, a method according to the invention is alsocharacterised in that the DNA sequence comprises or consists of thefollowing DNA sequence encoding OmpA and K2S or a functional variantthereof or a variant due to the degenerate nucleotide code:

(SEQ ID NO:2) ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCTCTGAGGGAAACAGTGACTGCTACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCCCACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATACCGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTGCAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCTCCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCCCTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGCTGTGGACAGAAGGATGTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGAC TGGATTCGTGACAACATGCGACCG

More preferably, a method according to the invention is alsocharacterised in that the DNA Sequence of OmpA comprises or consists ofthe following sequence or a functional variant thereof or a variant dueto the degenerate nucleotide code:

(SEQ ID NO:3) ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCC.

Said DNA encodes the following amino acid sequence of OmpA. OmpA thuscomprises or consists of a protein characterized by the following aminoacid sequence or a fragment, a functional variant, an allelic variant, asubunit, a chemical derivative or a glycosylation variant thereof aspart of the invention:

MKKTAIAIAVALAGFATVAQAA. (SEQ ID NO:21)

The untranslated region may contain a regulatory element, such as e.g. atranscription initiation unit (promoter) or enhancer. Said promoter may,for example, be a constitutive, inducible or development-controlledpromoter. Preferably, without ruling out other known promoters, theconstitutive promoters of the human Cytomegalovirus (CMV) and Roussarcoma virus (RSV), as well as the Simian virus 40 (SV40) and Herpessimplex promoter. Inducible promoters according to the inventioncomprise antibiotic-resistant promoters, heat-shock promoters,hormone-inducible “Mammary tumour virus promoter” and themetallothioneine promoter. Preferred promoters include the T3 promoter,T7 promoter, Lac/ara1 and Ltet0-1.

More preferably, a method according to the invention is alsocharacterised in that the DNA of the tPA, tPA variant, K2S molecule orK2S variant is preceded by a lac promoter and/or a ribosomal bindingsite such as the Shine-Dalgarno sequence (see also example).

More preferably, a method according to the invention is alsocharacterized in that the DNA coding for the tPA, tPA variant, K2Smolecule or K2S variant is selected from the group of DNA moleculescoding for at least 90% of the amino acids 87-527, 174-527, 180-527 or220-527 of the human tissue plasminogen activator protein (SEQ IDNO:19).

More preferably, a method according to the invention is alsocharacterised in that the DNA Sequence of K2S comprises or consists ofthe following sequence:

(SEQ ID NO:4) TCTGAGGGAAACAGTGACTGCTACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCCCACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATACCGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTGCAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCTCCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCCCTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGCTGTGGACAGAAGGATGTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCG ACCGTGA.

The present invention also relates to variants of the before-mentionednucleic acid molecules due to the degenerate code or to fragmentsthereof, nucleic acids which hybridize to said nucleic acids understringent conditions, allelic or functional variants. The invention alsorelates to nucleic acids comprising said K2S nucleic acid fused to thenucleic acid encoding another protein molecule.

Stringent conditions as understood by the skilled person are conditionswhich select for more than 85%, preferred more than 90% homology(Sambrook et al. 1989; Molecular Cloning: A Laboratory Manual, 2^(nd)ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Thehybridisation will be carried out e.g. in 6× SSC/5× Denhardt'ssolution/0.1% SDS (SDS: sodium dodecylsulfate) at 65° C. The degree ofstringency is decided in the washing step. Thus, for example for aselection of DNA-sequences with approx. 85% or more homology, theconditions 0.2×SSC/0.01% SDS/65° C. and for a selection of DNA-sequencesof approx. 90% or more homology the conditions 0.1×SSC/0.01% SDS/65° C.are suitable. The composition of said reagents is described in Sambrooket al. (1989, supra).

Another important part of the present invention is a variant of humantissue plasminogen activator comprising of or consisting of the Kringle2 (Barbas, C. F. III and Wagner, J., et al., Enzymology 8: 94-103(1995)) plus Serine protease (Bennett, W. F., et al., J. Biol. Chem.266:5191-5201 (1991)) (abbreviated K2S) protein or a variant or afragment, a functional variant, an allelic variant, a subunit, achemical derivative, a fusion protein or a glycosylation variantthereof.

The numbering/naming of the domains is according to Genbank accessionnumber GI 137119 or Nature 301 (5897), 214-221 (1983), wherein theKringle 2 domain extends from amino acid 176-262 and the protease domainfrom 276-527. Thus, according to the invention, a preferred K2S moleculemay include amino acids 176-527 including the amino acids betweenKringle 2 and the protease (amino acids 263 to 275; exemplified in FIG.8 (structure A)). A K2S molecule according to the invention comprisesthe minimal part of the Kringle 2 domain and the protease domain stillretaining protease activity and fibrin binding activity (measured asexemplified in the description/example). Said K2S molecule according tothe invention comprises the amino acids SEGN or SEGNSD in its N-terminalportion (see infra). A preferred K2S molecule does not include aminoacids 1 to 3 or 1 to 5 of the tPA molecule. Preferably, a K2S moleculeaccording to the invention has the amino acid Asn at positions 177 and184, i.e. it does not require the modifications as disclosed inWaldenström for improved producibility with a method according to theinvention. Thus, a preferred K2S molecule according to the invention hasthe native amino acid sequence (no mutation) as opposed to the moleculesknown from the prior art. Most preferred, said K2S molecule according tothe invention is a molecule characterized by the native amino acidsequence or parts thereof, does neither have amino acids 1 to 3 nor 1 to5 of tPA and comprises N-terminally the amino acids SEGN or SEGNSD forimproved producibility and/or correct folding of the molecule.

It is essential that the K2S protein according to the inventioncomprises in its N-terminal portion a peptide characterized by the aminoacid sequence SEGN which advantageously allows commercial productionwith a method as described supra leading to a correctly folded, secretedK2S protein. Said 4 amino acids characterized by SEGN may have one orseveral amino acids more N-terminal, however said amino acids have to belocated in the N-terminal portion as opposed to the C-terminal portion.Most preferably, said amino acids are located at the N-terminal portion.Preferably, the amino acids characterized by SEGN may be carry a pointmutation or may be substituted by a non-natural amino acid.

Thus, in another important embodiment the invention relates to a K2Sprotein characterized in that it comprises the amino acids defined bythe sequence SEGN or a variant or a fragment, a functional variant, anallelic variant, a subunit, a chemical derivative, a fusion protein or aglycosylation variant thereof.

Such fragments are exemplified e.g. in FIG. 10 (Structure B-1) and FIG.11 (Structure B-2) extending from amino acids 193-527. Structure B-1 hasthe native amino acid Cys in position 261, wherein in B-2 the amino acidis substituted by Ser. Further fragments according to the inventioncomprising the amino acids 220-527 (FIG. 14, structure C) or comprisingthe amino acids 260-527 (FIG. 15, structure D) may be modified accordingto the invention by addition of the amino acids SEGN (SEQ ID NO: 9)and/or substitution of Cys-261 by Ser. The artisan can determine theminimal length of a K2S molecule according to the invention in order toretain its biological function and generate a K2S molecule with improvedproducibility and/or correct folding by adding the amino acids SEGN (SEQID NO: 9) in the N-terminal portion. Thus, another preferred embodimentis said minimal K2S molecule with SEGN (SEQ ID NO: 9) at its N-terminalportion.

In another important embodiment the invention relates to a K2S proteincharacterized in that it comprises the amino acids defined by thesequence SEGNSD (SEQ ID NO: 10) or a variant or a fragment, a functionalvariant, an allelic variant, a subunit, a chemical derivative, a fusionprotein or a glycosylation variant thereof. Such fragments areexemplified e.g. in FIG. 12 (Structure B-3) and FIG. 13 (Structure B-4)extending from amino acids 191-527. Structure B-3 has the native aminoacid Cys in position 261, wherein in B-4 the amino acid is substitutedby Ser. Further fragments according to the invention comprising theamino acids 220-527 (FIG. 14, structure C) or comprising the amino acids260-527 (FIG. 15, structure D) may be modified according to theinvention by addition of the amino acids SEGNSD (SEQ ID NO: 10) and/orsubstitution of Cys-261 by Ser. The artisan can determine the minimallength of a K2S molecule according to the invention in order to retainits biological function and generate a K2S molecule with improvedproducibility and/or correct folding by adding the amino acids SEGNSD(SEQ ID NO: 10) in the N-terminal portion. Thus, another preferredembodiment is said minimal K2S molecule with SEGNSD (SEQ ID NO: 10) atits N-terminal portion.

Another more preferred embodiment of the present invention relates to aK2S protein comprising a protein characterized by the following aminoacid sequence or a variant or a fragment, a functional variant, anallelic variant, a subunit, a chemical derivative or a glycosylationvariant thereof:

(SEQ ID NO:11) SEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPG VYTKVTNYLDWIRDNMRP*.

According to the invention, * means STOP (i.e. encoded by a stop codon).This K2S molecule is exemplified in FIG. 8.

One variant of the K2S molecule according to the invention relates to afusion protein of K2S being fused to another protein molecule.

Another more preferred embodiment of the present invention relates to aK2S protein consisting of a protein characterized by the following aminoacid sequence:

(SEQ ID NO:11) SEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPG VYTKVTNYLDWIRDNMRP*.

Said K2S molecules may be encoded by a DNA molecule as described supra.

Another important aspect of the invention relates to a DNA moleculecharacterized in that it is coding for:

a) the OmpA protein or a functional derivative thereof operably linkedto

b) a DNA molecule coding for a polypeptide containing the kringle 2domain and the serine protease domain of tissue plasminogen activatorprotein.

More preferably, a DNA molecule according to the invention is alsocharacterised in that the DNA sequence comprises or consists of thefollowing DNA sequence encoding OmpA and K2S or a functional variantthereof or a variant due to the degenerate nucleotide code:

(SEQ ID NO:2) ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCTCTGAGGGAAACAGTGACTGCTACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCCCACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATACCGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTGCAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCTCCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCCCTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGCTGTGGACAGAAGGATGTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGAC TGGATTCGTGACAACATGCGACCG

Said DNA molecule encodes the following fusion protein of OmpA and K2S.Said fusion protein of OmpA and K2S characterised in that it comprisesor consists of a protein characterized by the following amino acidsequence or a fragment, a functional variant, an allelic variant, asubunit, a chemical derivative or a glycosylation variant thereof formsan important part of the present invention:

(SEQ ID NO:8) MKKTAIAIAVALAGFATVAQAASEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRPG

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence b)is coding for at least 90% of the amino acids 87-527 of the human tissueplasminogen activator protein (numbering used herein as GI 137119 orNature 301 (5897), 214-221 (1983).

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence b)is coding for at least 90% of the amino acids 174-527 of the humantissue plasminogen activator protein.

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence b)is coding for at least 90% of the amino acids 180-527 of the humantissue plasminogen activator protein.

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence b)is coding for at least 90% of the amino acids 220-527 of the humantissue plasminogen activator protein.

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence a)is hybridizing under stringent conditions to the following sequence:

(SEQ ID NO:3) ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCC.

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence a)consists of the following sequence:

(SEQ ID NO:3) ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCC.

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence b)is hybridizing under stringent conditions to the following sequence:

(SEQ ID NO:4) TCTGAGGGAAACAGTGACTGCTACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCCCACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATACCGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTGCAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCTCCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCCCTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGCTGTGGACAGAAGGATGTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCG ACCGTGA.

Another preferred aspect of the invention relates to a DNA moleculeaccording to the invention, characterized in that said DNA sequence b)consists of the following sequence:

(SEQ ID NO:4) TCTGAGGGAAACAGTGACTGCTACTTTGGGAATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCTCCTGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCCCAGGCACTGGGCCTGGGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCACCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCCCACTGCTTCCAGGAGAGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATACCGGGTGGTCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTGCAGCTGCCGGACTGGACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCTCCTTTCTATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCCCTGGTGTGTCTGAACGATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGCTGTGGACAGAAGGATGTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCG ACCGTGA.

Another preferred embodiment of the invention relates to a vectorcontaining a DNA sequence according to the invention.

Another preferred embodiment of the invention relates to a vectoraccording to the invention, wherein said DNA sequence is preceded by alac promoter and a ribosomal binding site. Suitable vectors according tothe invention include, but are not limited to viral vectors such as e.g.Vaccinia, Semliki-Forest-Virus and Adenovirus, phagemid vectors and thelike. Preferred are vectors which can be advantageously used in E. coli,but also in any other prokaryotic host such as pPROTet.E, pPROLar.A,members of the pBAD family, pSE family, pQE family and pCAL.

Another preferred embodiment of the invention relates to the vectorpComb3HSS containing a DNA according to the invention, wherein theexpression of the gp III protein is suppressed or inhibited by deletingthe DNA molecule encoding said gp III protein or by a stop codon betweenthe gene coding for a polypeptide containing the kringle 2 domain andthe serine protease domain of tissue plasminogen activator protein andthe protein III gene.

Another important aspect of the present invention relates to aprokaryotic host cell comprising a DNA molecule according to theinvention.

Another important aspect of the present invention relates to aprokaryotic host cell comprising a vector according to the invention.

Another important aspect of the present invention relates to an E. colihost cell comprising a DNA molecule according to the invention.

Another important aspect of the present invention relates to a E. colihost cell comprising a vector according to the invention.

Yet another important aspect of the present invention is the use of aDNA molecule according to the invention or of a vector according to theinvention or a host cell according to the invention in a method for theproduction of a polypeptide having the activity of tissue plasminogenactivator.

Yet another important aspect of the present invention is the useaccording the invention as described above, wherein said method is amethod according to the invention.

Another very important aspect is a pharmaceutical composition comprisinga substance obtainable by a method according to the invention andpharmaceutically acceptable excipients and carriers. An example for saidsubstance is the K2S molecule described supra. The term“pharmaceutically acceptable carrier” as used herein refers toconventional pharmaceutic excipients or additives used in thepharmaceutical manufacturing art. Such physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients (see also e.g. Remington's Pharmaceutical Sciences (1990,18th ed. Mack Publ., Easton)). Said pharmaceutical composition accordingto the invention can be advantageously administered intravenously as abolus, e.g. as a single bolus for 5 to 10 seconds intravenously.

The invention further relates to the use of substances obtainable by amethod according to the invention in the manufacture of a medicament inthe treatment of stroke, cardiac infarction, acute myocardialinfarction, pulmonary embolism, any artery occlusion such as coronaryartery occlusion, intracranial artery occlusion (e.g. arteries supplyingthe brain), peripherally occluded arteries, deep vein thrombosis orrelated diseases associated with unwanted blood clotting.

The following example is intended to aid the understanding of theinvention and should in no way be regarded as limiting the scope of theinvention.

EXAMPLES Example 1 Materials and Methods

Primer Design

In order to amplify a specific part of tPA gene, a pair of primersSK2/174 [5′ GAGGAGGAGGTGGCCCAGGCGGCCTCTGAGGGAAACAGTGAC 3′] (SEQ IDNO:22) and ASSP [5′ GAGGAGGAGCTGGCCGGCCTGGCCCGGTCGCATGTTGTCACG 3′] (SEQID NO:23) were synthesized (Life Technologies, Grand Island, N.Y.).These primers were designed based on the human tPA gene retrieved fromNCBI databases (g137119). They were synthesized with Sfi I end cloningsites (underlined) in such a way that the reading frame from the ATG ofthe gpIII gene in phagemid vector, pComb3HSS, will be maintainedthroughout the inserted sequence.

Another primer set for site-directed mutagenesis was designed to annealat the sequence situated between K2S gene and gene III in pComb3H-K2S.The sequence of primers with mutation bases (underlined) for generatinga new stop codon were MSTPA [5′ ACATGCGACCGTGACAGGCCGGCCAG 3′] (SEQ IDNO:24) and MASTPA [5′ CTGGCCGGCCTGTCACGGTCGCATGT 3′] (SEQ ID NO:25).

Amplification of K2S gene by PCR. One μg SK2/174 and ASSP primerstogether with 50 ng of p51-3 template (obtained from Dr. Hiroshi Sasaki,Fujisawa Pharmaceutical, Japan) were suspended in 100 μl PCR mixture. Anamount of 2.5 U Taq polymerase (Roche Molecular Biochemicals,Indianapolis, Ind.) was finally added to the solution. The titratedamplification condition was initiated with jump start at 85° C. for 4min, then denaturation at 95° C. for 50 sec, annealing at 42° C. for 50sec, extension at 72° C. for 1.5 min. Thirty five rounds were repeatedlyperformed. The mixture was further incubated at 72° C. for 10 min. Theamplified product of 1110 bp was subsequently purified by QIAquick PCRPurification Kit (QIAGEN, Hilden, Germany). The correctness of purifiedproduct was confirmed by restriction enzymes.

Construction of phagemid expressing K2S. The purified PCR product of K2Sand pComb3HSS phagemid (kindly provided by Dr. Carlos F. Barbas, ScrippsInstitute, USA) were digested with Sfi I (Roche Molecular Biochemicals,Indianapolis, Ind.) to prepare specific cohesive cloning sites. Four μgof the purified PCR product was digested with 60 U of Sfi I at 50° C.for 18 h. For pComb3HSS, 20 μg of phagemid vectors were treated with 100U of Sfi I. Digested products of purified PCR product of K2S andpComb3HSS (˜3300 bp) were subsequently gel-purified by the QIAquick GelExtraction Kit (QIAGEN, Hilden, Germany). T4 ligase (Roche MolecularBiochemicals, Indianapolis, Ind.) of 5 U were introduced to the mixtureof 0.7 μg of purified Sfi I-digested pComb3HSS and 0.9 μg of purifiedSfi I-digested PCR product. Ligation reaction was incubated at 30° C.for 18 h. The newly constructed phagemid was named pComb3H-K2S.

Transformation of E. coli XL-1 Blue. Two hundred μl of CaCl₂ competentE. coli XL-1 Blue (Stratagene, La Jolla, Calif.) were transformed with70 ng of ligated or mutated product. The transformed cells werepropagated by spreading on LB agar containing 100 μg/ml ampicillin and10 μg/ml tetracycline (Sigma, Saint Louis, Mo.). After cultivation at37° C. for 18 h several antibiotic resistant colonies were selected forplasmid minipreps by using the alkaline lysis method. Each purifiedplasmid was subjected to Sfi I restriction site analysis. A transformantharboring plasmid with the correct Sfi I restriction site(s) wassubsequently propagated for 18 h at 37° C. in 100 ml LB broth withampicillin 100 μg/ml and tetracycline 10 μg/ml. A plasmid maxiprep wasperformed using the QIAGEN Plasmid Maxi Kit (QIAGEN, Hilden, Germany).The purified plasmid was reexamined for specific restriction sites bySfi I and sequenced by AmpliTaq DNA Polymerase Terminator CycleSequencing Kit (The Perkin-Elmer Corporation, Forster City, Calif.).

Site-directed mutagenesis of pComb3H-K2S. 10 ng of pComb3H-K2S templatewere mixed with 125 ng of MSTPA and MASTPA primers. PfuTurbo DNApolymerase (Stratagene, LA Jolla, Calif.) of 2.5 U was added to themixture for cycle amplification. The reaction started with one round of95° C. for 30 sec. Then it was followed by 16 rounds consisting of 95°C. for 30 sec, 55° C. for 1 min, and 68° C. for 9 min. The reaction tubewas subsequently placed on ice for 2 min. In order to destroy thetemplate strands, 10 U of Dpn I restriction enzyme (Stratagene, LAJolla, Calif.) were added to the amplification reaction and incubatedfor 1 h at 37° C. This synthesized product (MpComb3H-K2S) was furtherused to transform E. coli XL-1 Blue.

Preparation of phage-display recombinant-K2S. After pComb3H-K2S wastransformed to XL-1 Blue, the phage display technique was performed. Aclone of pComb3H-K2S transformed E. coli XL-1 Blue was propagated in 10ml super broth containing ampicillin 100 μg/ml and tetracycline 10 μg/mlat 37° C. until the O.D. [600 nm] of 1.5 was reached. The bacterialculture was subsequently propagated in 100 ml of the same medium andculture for 2 h. An amount of 10¹² pfu of VCSM13 helper phage(Stratagene, La Jolla, Calif.) was used to infect the transformed E.coli XL-1 Blue. After 3 h incubation, kanamycin at a final concentrationof 70 μg/ml final concentration was added to culture. The culture wasleft shaking (200 RPM) for 18 h at 37° C. Bacteriophages which harboredK2S on gpIII (K2S-φ) were then harvested by adding 4% w/v PEG MW 8000(Sigma, Saint Louis, Mo.) and 3% w/v NaCl. Finally, the harvested phagewas resuspended in 2 ml PBS pH 7.4. The phage number was determined byinfecting E. coli XL-1 Blue. The colony-forming unit per milliliter(cfu/ml) was calculated as described previously (Lobel, L. I., et al.,Endocrinology. 138:1232-1239 (1997)).

Expression of recombinant-K2S in shaker flasks. MpComb3H-K2S transformedE. coli XL-1 Blue was cultivated in 100 ml super broth (3% w/v tryptone,2% w/v yeast extract and 1% w/v MOPS) at pH 7.0 in the presence ofampicillin (100 μg/ml) at 37° C. until an O.D. [600 nm] of 0.8 wasreached. Subsequently, the protein synthesis was induced by 1 mM of IPTG(Promega, Madison, Wis.). The bacteria were further cultured shaking(200 RPM) for 6 h at 30° C. The culture supernatant was collected andprecipitated with 55% saturated ammonium sulfate (Soeda, S., et al.,Life Sci. 39:1317-1324 (1986)). The precipitate was reconstituted withPBS, pH 7.2, and dialysed in the same buffer solution at 4° C. for 18 h.Periplasmic proteins from bacterial cells were extracted by using achloroform shock as previously described by Ames et al. (Ames, G. F., etal., J. Bacteriol. 160:1181-1183 (1984)).

Immunoassay quantification of recombinant-K2S. In order to detect r-K2S,solid phase was coated with monoclonal anti-kringle 2 domain (16/B)(generously provided by Dr. Ute Zacharias, Central Institute ofMolecular Biology, Berlin-Buch, Germany). The standard ELISA washing andblocking processes were preformed. Fifty μl of 10¹¹ cfu/ml of K2S-φ orsecretory r-K2S were added into each anti-kringle 2 coated well.Antigen-antibody detection was carried out as follows. Either sheepanti-M13 conjugated HRP (Pharmacia Biotech, Uppsala, Sweden) or sheepanti-tPA conjugated HRP (Cedarlane, Ontario, Canada), was added to eachreaction well after the washing step. The substrate TMB was subjected toevery well and the reaction was finally ceased with H₂SO₄ solution after30 min incubation. The standard melanoma tPA 86/670 (National Institutefor Biological Standards and Control, Hertfordshine, UK) was used aspositive control.

Amidolytic activity assay. A test kit for the detection of tPAamidolytic activity was purchased from Chromogenix (Molndal, Sweden).The substrate mixture containing plasminogen and S-2251 was used todetermine serine protease enzymatic activity. The dilution of 10⁻² ofeach ammonium precipitated sample was assayed with and withoutstimulator, human fibrinogen fragments. The assay procedure wasaccording to the COASET tPA manual.

SDS-PAGE and immunoblotting. The dialysed precipitate-product fromculture supernatant was further concentrated 10 fold with centricon 10(AMICON, Beverly, Mass.). The concentrated sample was subjected toprotein separation by SDS-PAGE, 15% resolving gel, in the reducingbuffer followed by electroblotting to nitrocellulose. The nitrocellulosewas then blocked with 4% skimmed milk for 2 hr. In order to detectr-K2S, a proper dilution of sheep anti-tPA conjugated HRP was applied tothe nitrocellulose. The immunoreactive band was visualized by asensitive detection system, Amplified Opti-4CN kit (BIORAD, Hercules,Calif.).

Copolymerized plasminogen polyacrylamide gel electrophoresis. An 11%resolving polyacrylamide gel was copolymerized with plasminogen andgelatin as previously described by Heussen et al. (Heussen, C. andDowdle, E. B., Anal. Biochem. 102:196-202 (1980)). The stacking gel wasprepared as 4% concentration without plasminogen and gelatin.Electrophoresis was performed at 4° C. at a constant current of 8 mA.The residual SDS in gel slab was removed after gentle shaking at roomtemperature for 1 h in 2.5% Triton X-100. Then the gel slab wasincubated in 0.1 M glycine-NaOH, pH 8.3, for 5 h at 37° C. Finally, thegel slab was stained and destained by standard Coomassie brilliant blue(R-250) dying system. The location of the peptide harboring enzymaticactivity was not stained by dye in contrast to blue-paint background.

Results

Construction of K2S gene carrying vector. From the vector p51-3 weamplified the kringle 2 plus ther serine protease portion of tPA (Ser¹⁷⁴in kringle 2 domain to Pro⁵²⁷ in the serine protease) using primersSK2/174 and ASSP. The amplified 1110 bp product was demonstrated byagarose gel electrophoresis (FIG. 1, lane 2) and was inserted intopComb3HSS phagemid by double Sfi I cleavage sites on 5′ and 3′ ends inthe correct reading frame. Thus a new vector, pComb3H-K2S, harboring theK2S was generated. In this vector K2S is flanked upstream by the OmpAsignal sequence and downstream by gpIII. The correct insertion of K2Swas verified both by restriction analysis with Sfi I (FIG. 2, lane 3),PCR-anaysis (demonstration of a single band at 1110 bp), and DNAsequencing. The schematic diagram of pComb3H-K2S map is given in FIG. 3.

Phage-displayed r-K2S. VCSM13 filamentous phage was used to infectpComb3H-K2S transformed E. coli XL-1 Blue, X[K2S]. VCSM13 was propagatedand incorporated the K2S-gpIII fusion protein during the viral packagingprocesses. The harvested recombinant phage (K2S-φ) gave a concentrationof 5.4×10¹¹ cfu/ml determined by reinfecting E. coli XL-1 Blue withPEG-precipitated phages. These recombinant phage particles were verifiedfor the expression of r-K2S by sandwich ELISA. The phage-boundheterologous K2S protein was recognized by the monoclonal anti-kringle 2antibody (16/B) by using sheep anti-tPA conjugated HRP antibodydetection system. The absorbance of this assay was 1.12±0.03 (Table 1).The amount of K2S detectable on 10¹² phage particles is equal to 336 ngof protein in relation to the standard melanoma tPA. In order tocorroborate that K2S-gpIII fusion protein was associated with phageparticles, sheep anti-tPA conjugated HRP antibody was substituted bysheep anti-M13 antibody conjugated HRP. This immuno-reaction exhibitedan absorbance of 1.89±0.07 (Table 1). In contrast, if the captureantibody was sheep anti-M13 antibody, extremely low K2S was observedwith sheep anti-tPA antibody conjugated HRP; the absorbance was only0.17±0.01 (Table 1). This suggested that only a minority of purifiedphage particles carried K2S-gpIII fusion protein. VCSM13 prepared fromnon-transformed XL-1 Blue was used as a negative control.

Construction of MpComb3H-K2S. We generated a stop codon between K2S andgpIII in pComb3H-K2S with the aid of the mutagenic primers (MSTPA andMASTPA) (FIG. 4). In order to enrich the newly synthesized and mutatedMpComb3H-K2S, the cycle amplification mixture was thoroughly digestedwith Dpn I to degrade the old dam methylated pComb3H-K2S template (Dpn Iprefers dam methylated DNA). After transforming of E. coli XL-1 Bluewith MpComb3H-K2S, a transformant XM[K2S] was selected for furtherstudy. As a consequence of bp substitution, one Sfi I cleavage siteclose to the 3′ end of K2S gene was lost after site-directedmutagenesis. A linear version of Sfi I cleaved MpComb3H-K2S was observedat 4319 bp without the appearance of inserted K2S gene fragment (FIG. 5,lane 3). Thus, the K2S gene encoding by MpComb3H-K2S was expressed innon-gpIII fusion form in XM[K2S].

Expression and purification of K2S. K2S expression in XM[K2S] wasinduced by IPTG. r-K2S was detectable by using ELISA both in theperiplasmic space and in the culture supernatant. The amount of theheterologous protein in each preparation was determined by sandwichELISA and related to the standard tPA. From 100 ml of the bacterialculture in shaker flask with the O.D. [600 nm] of 50, the periplasmicfraction yielded 1.38 μg of r-K2S (approximately 32%) whereas 2.96 μg ofr-K2S (approximately 68%) was obtained in the ammonium precipitatedculture supernatant. Sandwich ELISA was used to verify the PEGprecipitated phage from VCSM13 infected XM[K2S]. No r-K2S captured bymonoclonal anti-kringle 2 antibody was detected by anti-M13 conjugatedHRP, indicating that K2S is not presented on the phage particles ifgpIII is missing.

Amidolytic activity measurement. If serine protease domain is present inthe sample, plasminogen will be converted to plasmin. The producedplasmin will further digest the S-2251 substrate to a colour product,p-nitroaniline, which has a maximum absorbance at 405 nm. The specificactivity of the recombinant product is in accordance with theabsorbance. The fibrinogen-dependent enzymatic activity of each samplei.e. K2S-φ, periplasmic r-K2S or culture supernatant r-K2S, wasevaluated and compared. Both K2S-φ and periplasmic r-K2S illustratednotably low enzymatic activity, which was below the sensitivity of thetest (0.25 IU/ml). The culture supernatant r-K2S gave thefibrinogen-dependent enzymatic activity of 7 IU/ml. Thus, from 100 mlculture we obtained a total of 700 IU enzymatic activity. Withoutfibrinogen no enzymatic activity of the r-K2S purified from culturesupernatant was observed—whereas standard melanoma tPA showed someactivity.

Demonstration of recombinant protein by immunoblotting. Partiallypurified K2S from culture supernatant of XM[K2S] revealed a molecularmass of 39 kDa by using sheep anti-tPA antibodies (FIG. 6). The negativecontrol, partially purified culture supernatant of non-transformedXL1-Blue, contained no reactive band with a similar size.

Localization of active enzyme by PAGE. The plasminogen has beencopolymerized and immobilized with gelatin in the polyacrylamide gelprior to electrophoresis. The ammonium sulfate precipitated culturesupernatants of E. coli XL-1 Blue, E. coli XL-1 Blue transformed withpComb3HSS and XM[K2S] were analyzed (FIG. 7). All samples were processedin non-reducing condition to preserve the correct conformation andactivity of the serine protease domain. Transparent areas of serineprotease digested plasminogen were observed only in the ammonium sulfateprecipitated culture supernatants of XM[K2S] at 34 and 37 kDa positions.The other samples gave no clearing zones. The positive control lane ofstandard melanoma tPA also demonstrated enzymatic activity at 66 and 72kDa positions.

1. A fusion polypeptide comprising from N- to C-terminal: (a) an OmpAsignal peptide; (b) a peptide selected from the group consisting of SEGN(SEQ ID NO:9) and SEGNSD (SEQ ID NO:10); and (c) a peptide selected fromthe group consisting of tPA or K2S molecule.
 2. The fusion polypeptideof claim 1, wherein the OmpA signal peptide is encoded by the sequenceof SEQ ID NO:3.
 3. The fusion polypeptide of claim 1, wherein saidpeptide of (b) is SEGN (SEQ ID NO: 9) and is encoded by the sequenceTCTGAGGGAAAC (SEQ ID NO:20).
 4. The fusion polypeptide of claim 1,wherein said peptide of (b) is SEGNSD (SEQ ID NO: 10) and is encoded bythe sequence TCTGAGGGAAACAGTGAC (SEQ ID NO: 1).
 5. The fusionpolypeptide of claim 1, wherein said peptide of (c) is a K2S moleculethat is encoded by the sequence of SEQ ID NO:4.
 6. The fusionpolypeptide of claim 1, wherein said peptide of (c) is a K2S moleculeselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,and SEQ ID NO:18.
 7. The fusion polypeptide of claim 1, wherein saidpeptide of (c) is a tPA molecule having at least 90% identity to aminoacids 87-527 of SEQ ID NO:19.
 8. The fusion polypeptide of claim 1,wherein said peptide of (c) is a tPA molecule having at least 90%identity to amino acids 174-527 of SEQ ID NO:19.
 9. The fusionpolypeptide of claim 1, wherein said peptide of (c) is a tPA moleculehaving at least 90% identity to amino acids 180-527 of SEQ ID NO:19. 10.The fusion polypeptide of claim 1, wherein said peptide of (c) (c) is atPA molecule having at least 90% identity to amino acids 220-527 of SEQID NO:19.
 11. The fusion polypeptide of claim 1, wherein a DNA sequenceencoding said tPA of (c) hybridizes under stringent conditions to amolecule consisting of SEQ ID NO: 4, wherein the hybridization iscarried out in 6× SSC, 5× Deinhardt's solution, and 0.1% SDS at 65° C.followed by a washing step in 0.2 SSC, 0.01% SDS at 65° C.